1,4-Dihydropyridines useful for prevention or reduction of atherosclerotic lesions on arterial walls

ABSTRACT

The subject invention provides methods for preventing, retarding, or reducing atherosclerotic lesions or atherosclerotic degradation of aterial walls or treating atherosclerosis comprising administering to a mammal in need of such prevention, retardation, reduction or treatment at least one compound having the formula I: ##STR1## wherein: Ph is phenyl, 
     Ar is: 2-nitrophenyl, 3-nitrophenyl, 2,3-dichlorophenyl or benzofurazan-4-yl, 
     A is a branched chain alkylene radical having from 2 to 6 carbon atoms, 
     R is a straight or branched chain alkyl radical having from 1 to 6 carbon atoms, optionally mono-substituted by an alkoxy substituent having from 1 to 6 carbon atoms, 
     R 1  is hydrogen, hydroxy, or an alkyl radical having from 1 to 4 carbon atoms, 
     R 2  is hydrogen, or methyl; 
     or an enantiomer of a compound having formula I; a hydrated compound having formula I; a solvated compound having formula I; a pharmaceutically acceptable acid addition salt, or any of the foregoing.

FIELD OF THE INVENTION

The present invention relates to the use of 1,4-dihydropyridines tocounter several processes which affect the development ofatherosclerotic vascular lesions, such as, for example, myocyteproliferation and migration, cholesterol metabolism in macrophages andoxidative modification of low-density lipoproteins (LDL). Beneficialeffects on the above biological processes are the basis for preventionof atherosclerotic degradation in arterial walls of humans. Anotheraspect of the invention relates to particular forms of, and compositionscontaining, such compounds and adapted for this therapeutic use.

BACKGROUND OF THE INVENTION

The compounds of the invention are known for their coronary dilating andantihypertensive activity from U.S. Pat. No. 4,705,797. However, theanhydrous form of such compounds is novel and forms part of the presentinvention.

A particularly preferred compound is methyl1,1,N-trimethyl-N-(3,3-diphenylpropyl)-2-aminoethyl1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-pyridine-3,5-dicarboxylate(lercanidipine), and pharmaceutically acceptable salts thereof. Thesecompounds can be prepared according to the methods cited in U.S. Pat.No. 4,705,797 or the methods disclosed herein.

Also, particularly preferred are the resolved enantiomers oflercanidipine. The preparation of the individual enantiomers isdescribed in the present specification.

Arteriosclerosis, a generic term for thickening and hardening of thearterial wall, is responsible for many deaths in the United States andother westernized societies. One type of arteriosclerosis isatherosclerosis, a disorder of the larger arteries that underlies mostcoronary artery disease, aortic aneurysm, and arterial disease of thelower extremities. Atherosclerosis plays a major role in cerebrovasculardisease and is a leading cause of death in the United States, both aboveand below age 65 (E. L. Bierman in Harrison's Principles of InternalMedicine XII Ed., page 992 (1991)).

It is now recognized that atherosclerosis is a multifactorial processthat, when leading to clinical sequelae, is based on extensiveproliferation of migrated smooth muscle cells (myocytes) within theintima of the affected artery. The atherosclerotic plaque formation isbelieved to be the result of three fundamental biological processes.These are:

1) migration and proliferation of intimal smooth muscle cells, togetherwith variable numbers of accumulated macrophages and T-lymphocytes;

2) formation by the proliferated smooth muscle cells of large amounts ofconnective tissue matrix, including collagen, elastic fibers, andproteoglycans; and

3) accumulation of lipid, principally in the form of cholesterol estersand free cholesterol within the cells as well as in the surroundingconnective tissues.

(R. Ross et al., Science, 180:1332 (1973); R. Ross et al., N. Eng. J.Med., 295:369 (1976); R. W. Wissler et al, Prog. Cardiovasc. Dis.,18:341 (1976)).

In addition, a number of experimental reports ascribe a key role to theoxidative modification of low density lipoproteins (LDL) in the earlystages of atherosclerosis in humans, where hypercholesterolemiarepresents the major risk factor associated with the increased incidenceof the disease. Available data has lead to the belief that LDL canundergo oxidative modification (D. Steinberg et al., N. Eng. J. Med.,320:915 (1989); J. L. Witztum, Lancet, 344:793 (1994)) and that oxidizedLDL has been shown to promote atherogenesis by a number of mechanisms,including enhanced uptake of LDL in tissue macrophages which leads tolipid accumulation and chemotactic activity for monocytes (M. S. Brownet al, Ann. Rev. Biochem., 52:223 (1983)), and cytotoxicity to arterialwall endothelial cells (S. Parthasarathy et al., Prog. Lipid Res.,31:127 (1992)).

SUMMARY OF THE INVENTION

It has now been found that 1,4-dihydropyridines, such as lercanidipineand each enantiomer of lercanidipine are able to counteract many of thebiological processes leading to atherosclerotic lesions. Therefore,these compounds are useful in a mammal, including a human in need ofsuch treatment to prevent, retard, or reduce the atheroscleroticdegradation of the arterial wall, hypercholesterolemia and the variousdiseases caused by these, such as, for example, ischemic heart diseasessuch as myocardial infarction and cerebrovascular diseases such ascerebral infarction and cerebral apoplexy.

The compounds of the invention are also useful for the inhibition ofrestenosis following percutaneous transluminal coronary angioplasty(PTCA) and for suppressing the progression of vascular hypertrophyassociated with hypertension.

The subject invention provides a method for preventing, retarding,and/or reducing atherosclerotic lesions and preventing atheroscleroticdegradation of arterial walls. The method comprises administration of aneffective amount of a compound having Formula I: ##STR2## wherein:

Ph is phenyl,

Ar is: 2-nitrophenyl, 3-nitrophenyl, 2,3-dichlorophenyl orbenzofurazan-4-yl,

A is a branched chain alkylene radical having from 2 to 6 carbon atoms,

R is a straight or branched chain alkyl radical having from 1 to 6carbon atoms, optionally mono-substituted by an alkoxy substituenthaving from 1 to 6 carbon atoms,

R₁ is hydrogen, hydroxy, or an alkyl radical having from 1 to 4 carbonatoms, and

R₂ is hydrogen, or methyl.

Also useful in the present invention are resolved enantiomers of thecompounds having formula I, pharmaceutically acceptable acid additionsalts, and hydrated or solvated forms of any of these compounds.

The present invention also contemplates unit dosage forms that includean effective amount of at least one of the (R)-enantiomer, the(S)-enantiomer, or the racemate of the compounds of the invention.

It has been discovered that the compounds of the invention can beprepared in anhydrous (unsolvated) form by recrystallizing the compoundin an aprotic solvent and a protic solvent. In the recrystallizationeither aprotic or protic solvent can be used prior to the other. Theanhydrous compounds formed have improved stability when compared to thehydrated or solvated compounds. Particularly preferred among these1,4-dihydropyridine derivatives are lercanidipine, its enantiomers, andpharmaceutically acceptable salts thereof. Lercanidipine is methyl1,1-N-trimethyl-N-(3,3-diphenylpropyl)-2-aminoethyl1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-pyridine-3,5-dicarboxylate.

The (S)-enantiomer and the racemate of lercanidipine, both possessantihypertensive activity, and can be used for patients in need oftreatment for both hypertension and diseases related to atheroscleroticphenomena.

On the other hand, (R)-lercanidipine, having only minimalantihypertensive activity, has substantial activity in, and can be usedfor treating conditions involving, smooth muscle cell migration andproliferation and diseases related to atherosclerotic phenomena withoutany concomitant cardiovascular effect. This enantiomer is useful fortreatment of patients for whom reduction of blood pressure would beundesirable.

The invention also provides for the preparation of a composition usefulfor the treatment, i.e., the prevention, arrest, retardation, or atleast partial reversal of atherosclerotic degradation in the arterialwalls of a patient in need of said treatment using a compound of thegeneral formula I.

DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of the effect of lercanidipine andits enantiomers on ³ H!-thymidine incorporation into myocytes of ratsmooth muscle cells.

FIG. 2 is a graphical representation of the ability of lercanidipine andits enantiomers to interfere with the migration of arterial myocytes.

FIG. 3 is a graphical representation of the ability of lercanidipine andits enantiomers to inhibit the enzyme ACAT and cholesterolesterification induced by AcLDL in mouse peritoneal macrophage.

FIG. 4 is a graphical representation of the concentration-dependenteffect of lercanidipine and its enantiomers on cholesterolesterification in cholesterol ester loaded macrophages.

FIGS. 5 and 6 are graphical representations of the effect oflercanidipine and its enantiomers on cell-mediated oxidation of LDL.

FIG. 7 is a time course plot of cell-mediated oxidation and the effectsof lercanidipine after incubation.

DETAILED DESCRIPTION OF THE INVENTION

All patents, patent applications, and literature references cited in thespecification are hereby incorporated by reference in their entirety. Inthe case of inconsistencies, the present disclosure, includingdefinitions, will prevail.

"Treatment" of atherosclerosis is intended to include preventing,retarding, and/or reducing atherosclerotic lesions and preventing,retarding, and/or reducing atherosclerotic degradation of arterialwalls, in mammals, particularly humans. In particular, the inhibition orcounteracting of the following processes, which lead to atheroscleroticlesions, can be considered very useful for the treatment ofatherosclerosis. They are:

1) migration and proliferation of intimal smooth muscle cells, togetherwith variable numbers of accumulated macrophages and T-lymphocytes;

2) formation by the proliferated smooth muscle cells of large amounts ofconnective tissue matrix, including collagen, elastic fibers, andproteoglycans; and

3) accumulation of lipid, principally in the form of cholesteryl estersand free cholesterol within the cells as well as in the surroundingconnective tissues.

According to the present invention, treatment constitutes anyimprovement in one or more clinical or histological symptoms ordiagnostic markers observed by the attending physician or determined byquantitative or semiquantitative techniques. Non-limiting examplesinclude techniques such as, analysis of blood, ultrasound and otherimaging techniques such as angiography. Several of these techniques havebeen developed and are well-known. Patients who are likely to requirethis treatment are those suffering from or at risk for myocardialinfarct, stroke, hyperlipidemia, or hypertension.

Lercanidipine can be prepared by Hantzsch cyclization of methyl3-aminocrotonate (1) with1,1,N-trimethyl-N-(3,3-diphenylpropyl)-2-aminoethylα-acetyl-3-nitrocinnamate (2) (Scheme 1), according to the methoddescribed in U.S. Pat. No. 4,705,797. ##STR3##

However, the cyclisation method described in the U.S. Pat. No. 4,705,797leads to several impurities and consequently a low yield of the productis obtained. The removal of the reaction byproducts requires the use ofpurification techniques, e.g., column chromatography, which aredifficult to apply on an industrial scale.

Alternatively, lercanidipine can be prepared by esterification of1,4-dihydro-2,6-dimethyl-5-methoxycarbonyl-4-(3-nitrophenyl)pyridine-3-carboxylic acid (3) with2,N-dimethyl-N-(3,3-diphenylpropyl)-1-amino-2-propanol (4) (Scheme 2)according to the method described in Example 3. ##STR4##

Resolved lercanidipine enantiomers can be obtained by the esterificationmethod illustrated in Scheme 2 using the corresponding homochiral acidswhich, when present in 1:1 ratio, constitute the above cited racemicacid 3.

The homochiral acids, hereinbelow called for convenience, acid 5 (the(R)-enantiomer) and acid 6 (the (S)-enantiomer), can be convenientlyprepared by resolution of racemic acid 3 according to the methodreported by A. Ashimori et al., Chem. Pharm. Bull. 39:108 (1991).

The esterification reaction can be performed in the presence of acoupling agent (e.g.: dicyclohexylcarbodiimide, N,N'-carbonyldiimidazoleor diethyl cyanophosphonate), optionally in the presence of a promotingagent (e.g.: N-hydroxysuccinimide or 4-dimethylaminopyridine) in aproticor chlorinated solvents (e.g.: N,N-dimethylformamide or chloroform) attemperatures ranging from -10° to 140° C. according to well-knownsynthetic methods: Albertson, Org. React. Vol. 12, 205-212 (1962);Doherty et al, J. Med. Chem. 35:2 (1992); Ishihara, Chem. Pharm. Bull.39:3238 (1991).

Alternatively, lercanidipine enantiomers can be prepared by reactingacid 5 (or acid 6) with alkyl chloroformate in presence of a tertiaryamine (e.g., triethylamine), then adding the intermediate alcohol (4) atabout 0°-80° C. Optionally, a promoting agent (e.g.,1-hydroxypiperidine) may be added before the addition of theintermediate (4) (Albertson, Org. React. Vol. 12, 157 (1962)).

The compounds and enantiomers of the invention can also be prepared byconversion of acid 5 (or acid 6) into the corresponding acyl halideusing inorganic acid halides (e.g.: phosphorous pentachloride, oxalylchloride, phosphorous trichloride, phosphorous oxychloride, or thionylchloride) in a chlorinated solvent (e.g.: chloroform, dichloroethane,dichloromethane, 1,1,1-trichloroethane and the like), optionally in thepresence of promoting agents (e.g.: N,N-dimethylformamide) attemperature ranging from about -10° to about 85° C. The resolved acylhalides can optionally, but need not, be isolated before the addition ofthe intermediate alcohol (4).

The lercanidipine enantiomers thus obtained are purified according tomethods known in the art, either as a free base (e.g.: by columnchromatography) or as a salt (e.g.: by reprecipitation orrecrystallization). The above methods can also be used for all compoundshaving formula I.

It has been discovered that racemic mixtures of the compounds of theinvention purified by recrystallization can be prepared in anhydrous orunsolvated form by recrystallizing the compound from a solution of thecompound in; (i) an aprotic solvent; and (ii) a protic solvent. Theanhydrous compounds have improved stability and lower hygroscopicitywhen compared to the hydrated/solvated compounds.

The lercanidipine can be obtained by esterification from thecorresponding dihydropyridine acid in accordance with a one-stepreaction process starting with the acid chloride of compound 3, preparedin situ. This route provides improved yields and the formation of fewerreaction byproducts. The improved purity of the compounds of theinvention avoids the use of chromatographic columns to isolate thedesired final product. The compounds can be obtained directly bycrystallisation as the hydrochloride salt with a high degree of purity.

The lercanidipine hydrochloride can be prepared according to the presentinvention in the anhydrous crystalline form, melting within a two degreerange from about 185° to 190° C. range after recrystallisation of thecrude hydrochloride compound from a solution of the compound in; (i) anaprotic solvent; and (ii) a protic solvent.

Examples of aprotic solvents include chlorinated solvents, such as, forexample, chloroform, dichloromethane, dichloroethane, chlorobenzene,1,1,1-trichloroethane, and the like; non-chlorinated solvents, such as,for example, ethyl acetate, methyl acetate, tetrahydrofuran, dioxane,N,N-dimethyl-formamide, dimethylcarbonate, toluene, xylene, an (C₅ -C₇)alkane, a (C₅ -C₇) cycloalkane and the like.

Examples of protic solvents include solvents, such as, for example,methanol, ethanol, propanol, isopropanol, n-butanol, sec-butanol,tert-butanol and the like. All solvents can be used alone or optionallyin a mixture with other solvents. All water miscible solvents can beused alone or in a mixture which may comprise at least one solvent andoptionally include water.

Pharmaceutically acceptable salts are prepared from the free bases in aconventional manner.

Preferred pharmaceutically acceptable acid addition salts are those suchas, for example, hydrochloric, sulfuric, maleic, succinic, citric,methanesulfonic and toluenesulfonic acids, and the like.

The hydrated and solvated forms of the compounds of the inventioninclude compounds which have water or solvent molecules associated withthem. There can be as little as about 0.25 molecules of water or solventor there can be several water or solvent molecules for eachlercanidipine molecule.

Typical solvents which associate or solvate the lercanidipines aresolvents which can be used for recrystallization. Examples of thesesolvents include water, alcohols such as, for example, methanol,ethanol, propanol, isopropanol, n-butanol, sec-butanol, tert-butanol andthe like.

According to the invention, the 1,4-dihydropyridine derivative havingformula I may be administered to the patient as such, or in the form ofany of its pharmaceutically acceptable salts, hydrates or solvates. Ithas been determined that the bioavalibility and efficacy of thecompounds having formula I is comparable whether administered in theanhydrous form or in a hydrated or solvated form.

Preferred pharmaceutically acceptable acid addition salts include thoseformed with hydrochloric, sulfuric, maleic, succinic, citric,methanesulphonic and toluenesulphonic acids; they may be prepared fromthe free bases in conventional manner. Whatever the form (base, salt,hydrate or solvate), the active ingredient will usually be administeredin admixture with a pharmaceutically acceptable carrier.

For the purpose of oral therapeutic administration, the active compoundsof the invention can be incorporated with excipients and used in theform of tablets, troches, capsules, elixirs, suspensions, syrups,wafers, chewing gum, and the like.

These preparations will typically contain at least about 0.5% of activecompounds, but the amount of active ingredient can be varied dependingupon the particular form and may conveniently contain from about 5% toabout 70% of the weight of the unit. The amount of active compound insuch compositions is such that a suitable dosage will be obtained.However, the desired dosage can be obtained by administering a pluralityof dosage forms.

The compounds of the invention can be administered as an oral dosage offrom about 0.1 to about 400 mg, preferably from about 1 to about 200 mg,and most preferably from about 5 to about 100 mg, per subject treated,per day. Therapy can continue for several months or years orindefinitely.

Preferred compositions and preparations according to the invention areprepared so that an oral dosage unit form contains from about 0.1 toabout 400 mg of active compound.

The tablets, pills, capsules, troches, and the like can also contain,for example, the following optional ingredients: a binder such as, forexample, microcrystalline cellulose, tragacanth gum, gelatin, and thelike; an excipient such as, for example, starch or lactose, adisintegrating agent such as, for example, alginic acid, sodium starchglycolate, corn starch, and the like; a lubricant such as, for example,magnesium stearate or hydrogenated castor oil, a glidant such as, forexample, colloidal silicon dioxide. Sweetening agents such as, forexample, sucrose or saccharin or flavoring agents such as, for example,peppermint, methyl salicylate, or orange flavoring can also be added.

The active compounds of the invention can be orally administered, forexample, with an inert diluent or with an edible carrier. They can, forexample, be enclosed in gelatin capsules, or they can be compressed intotablets.

When the dosage unit form is a capsule, it can contain, in addition tomaterials described above, a liquid carrier such as, for example, afatty oil.

For the purpose of parenteral administration, the 1,4-dihydropyridinederivatives may be incorporated into a solution or suspension.

These preparations typically contain at least 0.1% of active compound,but the amount of active ingredient may vary between 0.5% and about 30%of the weight thereof. The amount of active compound in suchcompositions is such that a suitable dosage will be obtained.

Preferably a parenteral dosage unit contains between 0.5 to 100 mg ofactive compound. The solutions or suspensions may also include thefollowing components: a sterile diluent such as water for injection,saline solution, fixed oils, polyethylene glycols, glycerine, propyleneglycol or other synthetic solvents; an antibacterial agent such asbenzyl alcohol or methyl parabens; an antioxidant such as ascorbic acidor sodium bisulfite; a chelating agents such asethylenediaminetetraacetic acid; a buffer such as an acetate, a citrateor a phosphate and an agent for the adjustment of tonicity such assodium chloride or dextrose. Parenteral multiple dose vials may be ofglass or plastics material.

The parenteral multiple dose vials can be of glass or plastic materials.

Other dosage unit forms can contain various other materials which modifythe physical form of the dosage unit, for example, coatings. Thus,tablets or pills can be coated with a sugar, shellac, or another entericcoating agent.

A syrup can contain, in addition to the active compounds, sucrose as asweetening agent, preservatives, dyes, colorings, and flavorants.

For the purpose of parenteral therapeutic administration, the activecompounds of the invention can be incorporated into a physiologicallyacceptable solution or suspension.

These preparations preferably contain at least about 0.1% of activecompound, but may be varied to from about 0.5% to about 30% by weightthereof. The amount of active compound in these compositions is suchthat a suitable dosage of from about 5 to about 100 mg will be obtainedupon administration of the solution or suspension.

Preferred compositions and preparations according to the presentinventions are prepared so that a parenteral dosage unit contains fromabout 5 to about 100 mg of active compound.

The solutions or suspensions can also include the following components:a sterile diluent such as, for example, water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, propylene glycol,other synthetic solvents, and the like; antibacterial agents such as,for example, benzyl alcohol, methyl parabens, and the like; antioxidantssuch as, for example, ascorbic acid, sodium bisulfite, and the like;chelating agents such as, for example, ethylenediaminetetraacetic acidand the like; buffers such as, for example, acetates; citrates,phosphates, and the like, and agents for the adjustment of tonicity suchas, for example, sodium chloride, dextrose, and the like.

Materials used in preparing these various compositions for practicingthe invention should be pharmaceutically pure and non-toxic at the levelemployed.

Additional compositions suitable for administration by various routesand containing compounds according to the present invention are alsowithin the scope of the invention.

Dosage forms, additional ingredients and routes of administrationcontemplated herein include those disclosed in U.S. Pat. No. 4,089,969and U.S. Pat. No. 5,091,182, both incorporated by reference in theirentirety.

EXAMPLES

The following examples illustrate the invention without limitation.

Example 1 (S)-(+)-Methyl1,1-N-Trimethyl-N-(3,3-Diphenylpropyl)-2-Aminoethyl1,4-Dihydro-2,6-Dimethyl-4-(3-Nitrophenyl)pyridine-3,5-Dicarboxylate((S)-Lercanidipine) Hydrochloride.0.5 H₂ O

Thionyl chloride, 0.13 ml, was added, at -10° C., to a stirredsuspension of 0.54 g of(R)-1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-5-methoxycarbonylpyridine-3-carboxylicacid in 2.9 ml of anhydrous dichloromethane and 0.75 ml of anhydrousN,N-dimethylformamide under a nitrogen atmosphere, and sheltered fromthe direct light.

After 1 hour at 0° C., a solution of 0.48 g of2,N-dimethyl-N-(3,3-diphenyl-propyl)-1-amino-2-propanol, (prepared asdescribed in U.S. Pat. No. 4,705,797), in 1 ml of dichloromethane,cooled to -5° C., was added. After stirring for 3 hours at 0° C. andstanding overnight at 20°-25° C., the solvent was evaporated in vacuoand the residue was dissolved in 20 ml of ethyl acetate. The organicphase was washed sequentially, with brine (4 ml), 10% aqueous sodiumcarbonate solution (5×4 ml), brine (4 ml), 1N hydrochloric acid (5×5ml), brine (4 ml), 10% aqueous sodium carbonate solution (2×5 ml) andwith brine (4 ml). The organic phase was dried over anhydrous sodiumsulfate and evaporated to dryness in vacuo. The product was purified byflash chromatography on silica gel column eluting with petroleumether-acetone 85:15. The individual, pure, TLC fractions (petroleumether-acetone 7:3 by volume and chloroform-5N methanolic ammonia 99:1.1by volume) were combined and evaporated to provide a residue that wasdissolved in 75 ml of diethyl ether containing 3% of acetone. Afterfiltration the solution was acidified with 3N ethereal hydrogen chlorideand the precipitate was collected by suction filtration and dried at 78°C./15 mmHg to provide 0.66 g of the title compound.

M.P. 115°-125° C.; α!_(D) ²⁵ =+70.56° (MeOH; c=0.981).

Elemental analysis % for C₃₆ H₄₁ N₃ O₆.HCl.0.5 H₂ O Found: C, 65.47; H,6.57; N, 6.29; Cl, 5.32; H₂ O, 1.68 CaIc.: C, 65.79; H, 6.60; N, 6.39;Cl, 5.39; H₂ O, 1.37

¹ H-NMR Spectrum of the base at 200 MHz (CDCl₃, δ): 8.10 (m,1H)nitrophenyl, CH in 2 7.97 (m,1H) nitrophenyl, CH in 4 7.62 (m, 1H)nitrophenyl, CH in 6 7.33 (dd,1H) nitrophenyl, CH in 5 7.29-7.10 (m,10H) H benzhydryl aromatics 5.79 (bs,1H) pyridine, NH 5.05 (s, 1H)pyridine, CH in 4 3.92 (t,1H) benzhydryl, CH 3.63 (s, 3H) COOCH₃ 2.57(m, 2H) OC(CH₃)_(2CH) ₂ N 2.40-2.23 (m, 2H) N(CH₃)CH₂ CH₂ 2.33/2.27 (2s,6H) pyridine, CH₃ in pos. 2 and 6 2.19-2.09 (m, 2H) N(CH₃)CH_(2CH) ₂2.17 (s, 3H) NCH₃ 1.35/1.31 (2s 6H) OC(CH₃)₂ CH₂ N

Example 2 (R)-(-)Methyl1,1-N-Trimethyl-N-(3,3-Diphenylpropyl)-2-Aminoethyl1,4-Dihydro-2,6-Dimethyl-4-(3-Nitrophenyl)pyridine-3,5-Dicarboxylate((R)-Lercanidipine) Hydrochloride.H₂ O

The title compound was obtained by the method described in Example 1 forthe (S)-enantiomer, starting with(S)-1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-5-methoxycarbonylpyridine-3-carboxylicacid.

M.p. 115°-120° C., α!_(D) ²⁵ =-70.88° (MeOH, c=0.975).

Elemental analysis % for C₃₆ H₄₁ N₃ O₆.HCl.H₂ O Found: C, 64.93; H,6.62; N, 6.24; Cl, 5.41; H₂ O, 2.50 Calc.: C, 64.90; H, 6.60; N, 6.31;Cl, 5.32; H₂ O, 2.70

The ¹ H-NMR spectrum of the base in CDCl₃ was identical to the ¹ H-NMRspectrum for the (S)-enantiomer from Example 1.

Example 3 Methyl 1,1-N-Trimethyl-N-(3,3-Diphenylpropyl)-2-Aminoethyl1,4-Dihydro-2,6-Dimethyl-4-(3-Nitrophenyl) Pyridine-3,5-Dicarboxylate(Lercanidipine) Hydrochloride

Thionyl chloride, 45.8 g (0.385 mole), was added dropwise over 15minutes into a stirred mixture comprising 11 6.2 g (0.35 mole) of2,6-dimethyl-5-methoxycarbonyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylicacid (3) (prepared as described in German patent DE 2,847,237), 645 mLof anhydrous dichloromethane and 150 mL of anhydrous dimethylformamide,maintained under a nitrogen atmosphere at a temperature of -4° to +1° C.The mixture was stirred and maintained at this temperature for 1 hour.This was followed by dropwise addition of 104.1 g (0.35 mole) of2,N-dimethyl-N-(3,3-diphenyl-propyl)-1-amino-2-propanol (4) (prepared asdescribed in U.S. Pat. No. 4,705,797), dissolved in 105 mL of anhydrousdichloromethane, over 15 minutes at a temerature of -10° to 0° C. Afterstirring for 3 hours at 0° C. and standing overnight at roomtemperature, the solvent was evaporated under vacuum and the residuedissolved in 3500 mL of ethyl acetate. The organic solution was washedsequentially with: brine (700 mL), 10% aqueous sodium carbonate solution(5×700 mL), brine (700 mL), 1N hydrochloric acid (5×700 mL) and finallywith brine (700 mL). The organic layer was dried over anhydrous sodiumsulphate for 30 minutes, filtered, shaken with 23 g of charcoal andfiltered again. The solution volume was first reduced to about 1 literby evaporation under vacuum, and the product allowed to crystallize.After standing 24 hours at 0° to 5° C., the crystalline product wascollected by vacuum filtration and recrystallized from EtOH 99% toprovide 179.5 g (78% yield) of the title compound. M.P. 186°-188° C.

Example 4A Effects on Arterial Myocyte Proliferation

Animal models of vascular injury have shown that an arterial lesion isfollowed by proliferation of medial myocytes, many of which migrate intothe intima and further proliferate to form a neointimal lesion. Thecauses of these events are not completely understood. Recent findingshave concluded that myocytes make up approximately 90-95% of thecellular population of the atherosclerotic lesion in young adults andcompose an average of 50% of the advanced atherosclerotic plaque. Inaddition, vascular myocytes contribute to lesion formation and growth bysynthesis of an extracellular matrix or they can accumulate lipids andbecome foam cells.

Thus, the elucidation of the factors affecting these phenomena affordsnew entry points for selective interference and inhibition of theprocess of atherogenesis (R. W. Wissler et al:, Am. J. Med. 91:(S1B), 3S(1991); S. M. Schwartz et al., Circ. Res. 58:427 (1986); A. W. Cloves etal., J. Cardiov. Pharmacol. 14:512 (1989); R. W. Wissler et al., Ann. NYAcad. Sci. 598:418 (1990).

Migration of myocytes was investigated for the present invention usingrat aortic smooth muscle cells in the presence of fibrinogen aschemotactic factor, whereas for studies on their proliferation rat andhuman cells were used. Cell count and 3H!-thymidine incorporation wereused to evaluate myocytes growth. The methodology was as follows:

Myocytes were cultured from the intima-medial layer of the aortas ofmale Sprague-Dawley rats (200-250 g). Cells were grown in monolayers at37° C. in a humidified atmosphere of 5% CO₂ in Eagle's minimum essentialmedium supplemented with 10% (v/v) fetal calf serum, 100 U/mlpenicillin, 0.1 mg/ml streptomycin, 20 mM tricine buffer and 1% (v/v)non-essential amino acid solution. The medium was changed every thirdday. Cells were used between the 4th and 10th passage. Cell viabilitywas timely assessed by trypan blue exclusion. Myocytes were identifiedfor growth behavior, morphology and using monoclonal antibody specificfor α-actin, the actin isoform typical of myocytes. Human vascularmyocytes (A 617 from human femoral artery) kindly provided by Prof. G.Gabbiani of The General Pathology Institute (GenevaUniversity--Switzerland) were grown in the same culture conditions.(These or other human vascular myocytes can be obtained and cultured asdescribed by Corsini et al, Atherosclerosis, 101:117-125, (1993);Corsini et al, Pharm. Res, 23:173 (1991); Ross, R, J. Cell Bio., 50:172,(1971); and Skalli, O. et al, J. Cell Bio., 103:2787 (1986)).

Cells were seeded at various densities for rat (2×10⁵) and human (5×10⁴)myocytes/petri dish (35 mm), and incubated with Eagle's minimumessential medium supplemented with 10% fetal calf serum. Twenty-fourhours later the medium was changed to one containing 0.4% fetal calfserum to stop cell growth, and the cultures were incubated for 72 hours.At this time (time 0) the medium was replaced by one containing 10%fetal calf serum in the presence or absence of known concentrations ofthe 1,4-dihydropyridine compounds being tested (shown in Table 1). Theincubation was continued for an additional 72 hours at 37° C. Justbefore the addition of the test substances, an aliquot from each petridish was used for cell counting. Cell proliferation was evaluated bycell count after trypsinization of the monolayers using a CoulterCounter model ZM. Cell viability was assessed by trypan blue exclusion,and found to be greater than 95% at the drug concentrations used.

                  TABLE 1                                                         ______________________________________                                        MYOCYTES GROWTH INHIBITION MEASURED BY                                        CELL COUNT.                                                                   Myocytes                                                                             LE       (R)-LE   (S)-LE NI     LA                                     from   IC.sub.50 (μM)                                                                      IC.sub.50 (μM)                                                                      IC.sub.50 (μM)                                                                    IC.sub.50 (μM)                                                                    IC.sub.50 (μM)                      ______________________________________                                        SD Rat 31.2     30.7     33.3   38.8   23.6                                   SHR Rat                                                                              14.1      9.2     16.8   n.t.   n.t.                                   WK Rat 16.4     15.3     20.7   n.t.   n.t.                                   Human  25.3     n.t.     n.t.   n.t.   n.t.                                   ______________________________________                                         LE = Lercanidipine   SD = Sprague Dawley                                      NI = Nicardipine    SHR = Spontaneously Hypertensive                          LA = Lacidipine     WK = Wistar Kyoto                                         IC.sub.50 = concentration required to inhibit cell growth by 50%              n.t. = not tested                                                        

The data from Table 1 show that compounds of the invention,particularly, racemic lercanidipine and its resolved enantiomersinhibited the rat and human myocyte proliferation in aconcentration-dependent manner and showed practically the same activityas the reference 1,4-dihydropyridines tested (NI and LA).

It was found that lercanidipine (and its enantiomers) proved active onthe cells from all the species investigated, in particular, humans.

Example 4B Effects on Arterial Myocyte Proliferation

In another experiment, synchronization of myocytes to the G_(o) /G₁interphase of the cell cycle was accomplished by incubatinglogarithmically growing cultures (3×10⁵ cells/plate) for 96-120 hours ina medium containing 0.4% fetal calf serum. Quiescent cells wereincubated for 20 hours in a fresh medium with 10% fetal calf serum inthe presence of the tested drugs. Cell proliferation was then estimatedby nuclear incorporation of ³ H!thymidine, incubated with cells (1μCi/ml medium) for 2 hours. Radioactivity was measured with Filter-Countscintillation cocktail (AQUASOL® Scintillation cocktail available fromPackard-Groningen, Netherlands).

The results, expressed as cell proliferation (as % of control) againstamount of the compound of the invention (Lercanidipine and itsenantiomers) tested, are shown in FIG. 1. The decrease in ³ H!-thymidineuptake of myocytes confirmed the high potency of the compounds of theinvention in inhibiting cell proliferation.

Example 5 Effects on Arterial Myocyte Migration

Migration of rat myocytes was examined using a 48-well micro chemotaxischamber (Neuro-Probe, U.S.A.). Freshly trypsinized myocytes preparedaccording to the procedure in Example 4A were suspended in a mediumsupplemented with 5% fetal calf serum (assay medium). The lower wells,containing 27 μl of the assay medium that included fibrinogen (600μg/ml) as chemotactic agent, were covered with apolyvinylpyrrolidone-free polycarbonate filter (8 μM pore size). Fiftyμl of the cell suspension (1×10⁶ cells/ml) were placed in the uppercompartment with the tested compounds. Incubation was carried out for 5hours at 37° C. in an atmosphere of 95% air and 5% CO₂.

After incubation the filter was removed from the chamber andnon-migrated cells were scraped from the upper surface, and the filterswashed with phosphate buffered saline three times. The filters werestained with Diff-Quik (Merz-Dade AG, Switzerland). The number ofmyocytes per 100×high power field that had migrated to the lower surfaceof the filters was determined microscopically. Six high power fieldswere counted per sample and the results were averaged.

The results, expressed as migration of cells (as % of control) againstamount of the compound of the invention (Lercanidipine and itsenantiomers) tested, are shown in FIG. 2. These data demonstrate theability of lercanidipine, and its enantiomers to interfere with themigration of arterial myocytes. All the tested compounds were able toinhibit myocytes migration in a dose-dependent manner with the(R)-enantiomer showing the most pronounced effect.

Example 6A Effects on Cholesterol Metabolism in Mouse PeritonealMacrophages

Atheromas contain two main cell types, macrophages and smooth musclecells (R. Ross, Nature 362:801 (1993)). Macrophages are derived fromcirculating monocytes and are the main lipid-loaded cells in thelesions. The mechanism by which they accumulate lipoprotein cholesteroland develop into foam cells depends mainly upon receptor-mediatedprocesses, involving the so called "scavenger receptor" that recognizeschemically and biologically modified LDL, such as acetyl LDL (AcLDL) andoxidized LDL (Y. Kurihara et al., Curr. Opin. Lipidol 2:295 (1991)). Thescavenger receptor, unlike the LDL receptor, is not subject to feed-backregulation and the result is a massive accumulation of cholesterol infoam cells. Cholesterol accumulates in macrophages in esterified form bya process involving the enzyme acylcoenzyme A-cholesterolacyltransferase (ACAT) which catalyzes the cholesterol esterification incytoplasm (M. S. Brown et al, J. Biol. Chem. 255:9344 (1980)). Only freecholesterol, i.e., unesterified cholesterol can be removed frommacrophages.

Cholesterol esterification induced by AcLDL in mouse peritonealmacrophages was investigated as follows. Mouse peritoneal macrophageswere obtained by peritoneal lavage from mice (Balb/c Charles River,Calco, Italy) three days after intraperitoneal injection ofthioglycolate. Cells (2-3×10⁶) were plated in 35 mm wells withDulbecco's minimum essential medium containing 10% fetal bovine serum.After 3 hours, the dishes were washed to eliminate unattached cells andmaintained in Dulbecco's minimum essential medium plus 10% fetal bovineserum for 24 hours before use. After cell plating, experiments wereperformed at 37° C. in serum free Dulbecco's minimum essential mediumcontaining 0.2% essentially fatty acid-free bovine serum albumin, AcLDL,a specific ACAT inhibitor, S-58035 (See C. A. Ross et al., J. Biol,Chem. 259:815-819 (1984)), and lercanidipine, or the lercanidipineenantiomers indicated. Human LDL (d=1.019-1.063 g/ml) were isolated fromplasma of healthy volunteers by sequential ultracentrifugation (BeckmanL5-50, Palo Alto, Calif.). For acetylation, LDL were dialyzed against 0.15M NaCl , pH 7.4, diluted with an equal volume of saturated sodiumacetate and treated with acetic anhydride. For ¹²⁵ l!AcLDL, lipoproteinswere labelled with sodium ¹²⁵ l!iodide desalted by gel filtration onSephadex G-25 eluted with phosphate buffered saline. Specific activitywas 100-200 cpm/ng of protein. Trichloroacetic acid non-precipitableradioactivity was below 2% of total. All lipoproteins were sterilefiltered. The cells were incubated for 24 hours with the testedcompounds. After substitution of the medium with fresh solution,incubation was continued for additional 24 hours. During this secondincubation ¹²⁵ l!AcLDL was added (50 μg/ml). Cholesterol esterificationwas measured after addition of 1-¹⁴ C! oleic acid (0.68 mCi sample)complexed with bovine serum albumin during the last 1 or 2 hours ofincubation by subsequent determination of radioactivity associated withcellular cholesteryl esters.

The results, expressed as level of inhibition (as % of control) againstamount of the compound of the invention (Lercanidipine and itsenantiomers) tested, are shown in FIG. 3. The compounds of theinvention, particularly, lercanidipine and its enantiomers inhibited, ina concentration dependent manner, up to 90% of the formation ofesterified cholesterol (in other words the compounds of the inventioninhibited the esterifying effect of enzyme ACAT) induced by AcLDL inmouse peritoneal macrophage. The IC₅₀ values for lercanidipine and theenantiomers ranged from 8 to 15 μM, as shown in FIG. 3. The(R)-enantiomer was the most potent compound.

Example 6B Effects on Cholesterol Metabolism in Mouse PeriotnealMacrophages

Another set of experiments was performed to evaluate the effect oflercanidipine on cholesterol esterification in macrophages loaded withcholesterol ester before the addition of the compounds of the invention,this condition being the same as in foam cells.

Cells, prepared according to the procedure in Example 6A, are loadedwith cholesteryl esters by exposure for 24 hours to a medium containing50 μg/ml acetyl LDL. ACAT inhibition was then determined as in Example6A.

The results, expressed as level of inhibition (as % of control) againstamount of the compound of the invention (Lercanidipine and itsenantiomers) tested, are shown in FIG. 4. The compounds of theinvention, particularly, lercanidipine and its enantiomers inhibited, ina concentration dependent manner, up to 70% of the formation ofesterified cholesterol induced by AcLDL in mouse peritoneal macrophage.The lC₅₀ value for the (R)-enantiomer was about 7 μM. The (R)-enantiomerof lercanidipine was slightly more potent than the racemate and(S)-lercanidipine was the least potent among the compounds tested.

Example 7 Cholesteryl Ester Hydrolysis

It was shown that lercanidipine and its enantiomers at a 5 μMconcentration did not impair the capability of the macrophages tohydrolyze the esterified cholesterol stored in the cytoplasm. Theseexperiments were performed by incubating cells preloaded with ³H!cholesterol in the presence of the specific ACAT inhibitor S-58035.The blockage of intracellular reesterification of cholesterol allowedthe assessment of the ability of cells to hydrolyze the accumulatedcholesterol esters.

For studies involving the quantitation of the hydrolysis of cholesterylesters in cytoplasmic lipid droplets, cells, prepared according to theprocedure in Example 6A, are loaded with cholesteryl esters by exposurefor 24 hours to the medium containing 50 μg/mI acetyl LDL. 1,2-³H!Cholesterol is included in all loading media at a concentration of 0.5μCi/ml. After 24 hours loading period, during which time theradiolabeled cholesterol is incorporated and esterified, the cellmonolayers are washed and incubated an additional 24 hours in mediumcontaining 0.1% bovine serum albumin to allow the intracellular pools oflabelled cholesterol to equilibrate to the same specific activity. Toquantitate cholesteryl ester hydrolysis, the loaded cells are incubatedfor up to 24 hours in Dulbecco's minimum essential medium containing,0.1% bovine serum albumin, lercanidipines, and the compound S-58035, aninhibitor of acyl-coenzyme A-cholesterol acyltransferase. The inhibitionof acyl-coenzyme A-cholesterol acyltransferase prevents thereesterification of any free cholesterol generated by cholesteryl esterhydrolysis and thus allows assessment of the activity of the hydrolase.The hydrolysis of the cholesteryl esters is quantified by determiningthe decrease of radiolabeled cholesteryl esters (E. H. Harrison et al.,J. Lipid. Res. 31:2187 were washed with phosphate buffered saline andextracted with hexane:isopropanol (3:2 v/v). The media were extractedwith chloroform:methanol (2:1 v/v). After solvent removal, free andesterified cholesterol were separated by TLC (isooctane:diethylether:acetic acid, 75:25:2 by volume). Cholesterol mass or radioactivityof the spots were determined by an enzymatic method (BoehringerMannheim, Germany) (F. Bernini et al., Atherosclerosis 104:19 (1993)) orby liquid scintillation counting (Lipoluma Lumac, Landgraf, TheNetherlands) respectively.

The results are given in Table 2. These data show that the addition oflercanidipine and its enantiomers did not influence the cellularhydrolytic activity, documented by the values of radioactivity in theesterified cholesterol fraction. The compounds of the invention do notimpair the capability of the macrophages to hydrolyze the esterifiedcholesterol stored in the cytoplasm.

                  TABLE 2                                                         ______________________________________                                        EFFECT OF LERCANIDIPINE AND ITS ENANTIOMERS ON                                CHOLESTERYL ESTER HYDROLYSIS IN MACROPHAGES                                                            % of choles-                                                                  teryl ester                                          ______________________________________                                        AcLDL 50 μg/ml          31 ± 0.8                                        AcLDL 50 μg/ml + S-58035 1 μg/ml                                                                   15 ± 0.5                                        AcLDL 50 μg/ml + S-58035 1 μg/ml + 5 × 10.sup.-6 M                                           12 ± 1.2                                        AcLDL 50 μg/ml + S-58035 1 μg/ml + 5 × 10.sup.-6 M                                           14 ± 0.4                                        AcLDL 50 μg/ml + S-58035 1 μg/ml + 5 × 10.sup.-6 M                                           16 ± 2.1                                        ______________________________________                                         *LE = lercanidipine                                                      

Example 8 Effects of Lercanidipine on Chemical Oxidation of LDL

Experimental reports ascribe a key role for the oxidative modificationof LDL in the early stages of atherosclerosis in humans. These reportssuggest that LDL undergoes oxidative modifications in vivo (D. Steinberget al., N. Eng. J. Med. 320:915 (1989); D. Steinberg etal., JAMA264:3047 (1990); D. Steinberg, Circulation 84:1420 (1991); S.Yla-Herttuala, Ann. Med. 23:561 (1991); U.P. Steinbrecher, Curr. Opin.Lipidol. 1:411 (1990); J. L. Witztum, Lancet 344:793 (1994)) and thatoxidatively modified LDL (OX-LDL) may induce atherogenesis by a numberof mechanisms, including its enhanced uptake in tissue macrophages (viathe scavenger receptor pathway) which leads to lipid accumulation, andchemotactic activity for monocytes, and cytotoxicity to arterial wallendothelial cells (S. Parthasarathy et al, Prog. Lipid. Res. 31:127(1992).

The ability of Lercanidipine to function as an antioxidant was evaluatedby incubating LDL, isolated from human plasma, for 22 hours with anoxidizing agent (20 μM Cu⁺⁺) in the presence of different concentrationsof the test compounds (0.01 μM-50 μM). The LDL oxidation was followed bymonitoring conjugated diene formation with a ultraviolet (UV)spectrometer at 234 nm.

The experimental conditions were as follows. LDL (d=1.019-1.063) wereisolated from human pooled plasma by sequential ultracentrifugation at4° C. and 40,000 rpm in a 50 Ti rotor, using a L5-50 ultracentrifuge(Beckman, Palo Alto, Calif.). The LDL were then dialyzed against 0.15MNaCl containing 0.01% ethylenediaminetetraacetic acid pH 7.4, sterilizedby filtration through a 0.2 μM millipore filter and stored at 4° C.under nitrogen in the dark until use (up to 3 weeks). Before use, LDLwere dialyzed against ethylenediaminetetraacetic acid free phosphatebuffered saline, pH 7.4, on Sephadex G-25 columns (PD-10, Pharmacia FineChemicals, Uppsala, Sweden). Then the LDL were filtered through asterile 0.22 μM filter. In a phosphate buffered saline (50 μglipoprotein protein/ml), the LDL were oxidized by incubation at 25° C.with 20 μM CuSO₄ for 3 hours. The lercanidipine solution was prepared asa 10⁻² M stock solution in methanol or ethanol and added as a methanolor ethanol solution (maximum 1% v/v) prior to addition of the coppersolution. The effect of lercanidipine on the oxidation of LDL wasdetermined by continuous monitoring of the formation of conjugateddienes by recording the increase in the absorbance at 234 nm at 5 minintervals duri a three hour period, against a phosphate buffered salineblank, using a UV spectrophotometer (Beckman DU 640) equipped with acontinuous reading and an automatic 6-cell changer. The time prior tothe onset of oxidation (lag time) was calculated as the interceptbetween the line of the maximum slope of the propagation phase and thex-axis. The results are shown in Table 3.

                  TABLE 3                                                         ______________________________________                                        EFFECT OF LERCANIDIPINE ON THE LAG                                            TIME OF LDL OXIDATION                                                         Lercanidipine μM                                                                          Lag time                                                       ______________________________________                                        0.0            46.5 ± 4.6                                                  0.5            45.2 ± 3.7                                                  1.0            49.8 ± 4.7                                                  2.5             53.6 ± 3.3*                                                5.0             73.2 ± 4.8**                                               10.0            112.7 ± 5.2**                                              ______________________________________                                         *P < 0.05                                                                     **P < 0.01                                                               

As shown in Table 3, lercanidipine, at a concentration of 10 μM clearlyincreased the lag time for LDL oxidation, the time required beforeoxidation begins, more than 2 times. The data show that the effect ofthe racemate was concentration-dependent, with concentrations greaterthan or equal to 2.5 μM significantly suppressing the onset of LDLoxidation. The activity of the resolved enantiomers was comparable tothat of the racemate.

Example 9A Effects of Lercanidipine on Cell-mediated Oxidation

The antioxidant capacity of the compounds of the invention, oncell-mediated oxidation, was evaluated by incubating LDL and mousemonocyte-macrophage cells (ATCC TIB 67 J774A.1) with CuSO₄ solution inthe presence of apolipoprotein B (Apo B), lercanidipine or one of itsenantiomers at several concentrations. The inhibition of formation ofaldehydic breakdown products, i.e., secondary metabolites of lipidperoxidation, malonic dialdehyde (MDA), was assessed by detection ofdialdehyde formation using the thiobarbituric acid assay (A. N. Hanna etal., Biochem. Pharmacol. 45:753 (1993)).

The antioxidant capacity was evaluated by incubatingethylene-diaminetetraacetic acid free-LDL under sterile conditions with5 μM Cu⁺⁺ (100 μg Apo B/ml) in the presence of J774A.1 cells. Oxidationwas stopped after 22 hours incubation by addition of butylatedhydroxytoluene to the medium (final concentration 40 μM) dissolved inethanol. To the incubated sample (0.250 ml), was added trichloroaceticacid (0.750 ml, 0.20% w/v) followed by of thiobarbituric acid (0.750 ml,0.67% w/v). The samples were heated at 100° C. for 20 min, followed bycooling and centrifugation. Malonic dialdehyde equivalents werecalculated using 1,1,3,3-tetramethoxypropane as the standard.

The results, expressed as millimoles of MDA produced against amount ofthe compound of the invention (Lercanidipine and its enantiomers)tested, are shown in FIG. 5. The results show that lercanidipine and itsenantiomers were effective in reducing LDL oxidation.

Example 9B Effects of Lercanidipine on Cell-mediated Oxidation

Following the procedure in Example 9A, the effects of lercanidipine oncell-mediated oxidation were studied with a different cell line whichshares many of the properties of endothelial cells (EAhy-926). (Thecells were provided by: Prof. A. Catapano, Pharmacology Dept.,Universita Statale--Milano.) (These or other endothelial cells can beobtained as described by C-J. S. Edgall et al, Proc. Nat. AcAd. Sci.,80:3734-3737, (1983); and Gimbrone, M., Prog. Hemostasis Thromb.,3:1-28, (1976)). The cells were treated with lercanidipine at aconcentration of from about 10 to about 100 μM.

The results, expressed as millimoles of MDA produced against amount ofLercanidipine, are shown in FIG. 6. The results show that lercanidipinereduced the oxidation of LDL at a concentration of 10 to 100 μM.

Example 9C Effects of Lercanidipine on Cell-mediated Oxidation

In experiments 9A and 9B, the cell-mediated oxidation effects oflercanidipine were investigated after incubation for 22 hours. In orderto investigate the lower potency shown by lercanidipine in theseconditions, the extent of lipid peroxidation in its presence was studiedover time. A reaction mixture was prepared according to the procedure inExample 9A,using lercanidipine at a concentration of 30 μM. Samples werewithdrawn from the incubation medium and the oxidation product, MDA,measured as described above.

The results, expressed as millimoles of MDA produced against time areshown in FIG. 7. It can be seen that at a concentration of 30 μM,lercanidipine exerted a very high inhibition of lipid oxidation after 10hours incubation. This shows that lercanidipine, administered at anappropriate time is as effective on cell mediated LDL oxidation as it ison Cu⁺⁺ -mediated oxidation.

Lercanidipine proved the most potent 1,4-dihydropyridine tested in theassays herein, its potency being one order of magnitude higher than thatof lacidipine, the most potent of the 1,4-dihydropyridine compoundspreviously known to have this activity.

Example 10 Effects on Blood Pressure in Hypertensive Dogs

The antihypertensive effects of oral administration of lercanidipine andits enantiomers were tested in renal hypertensive dogs.

Male Beagle dogs weighing 12-13 kg, aging 1-3 years (Nossan Allevamenti,Italy) were used. Chronic sustained hypertension was induced bybilateral renal artery constriction, according to the Goldblatt method"two-kidney, two clip hypertension". Under barbiturate anaesthesia (35mg/kg i.v.), during two different surgical interventions one month apartfrom each other, both renal arteries were clipped with original renalsilver clips and narrowed by about 60-70%. After two months from thelast intervention, an experimental renal hypertension was produced andthe animals were suitable for the implantation of a catheter. Undersodium pentobarbital anaesthesia (35 mg/kg i.v.), in sterile conditions,the dogs were catheterized by inserting an indwelling cannula (PE 200Clay Adams) into the ascending aorta through the right common carotidartery. The catheter was subcutaneously exteriorized at the back of theneck, filled with heparinized saline solution and flushed daily toprevent clotting. After a week recovery time from surgery, the animalswere connected to a HP 1290A pressure transducer connected to an HP8805B carrier amplifier of a Hewlett Packard HP 7700 multichannelpolygraph, to monitor the arterial blood pressure. Heart rate wasmanually computed from the pressure trace.

All animals were alternatively treated with placebo, lercanidipine andits (R)- and (S)- enantiomers. The drugs were administered orally with astraight round aseptic tip catheter (Pores Serlat--France). The drugswere suspended in aqueous 0.5% Methocel A4C plus Antifoam M10 (10%). Theamount administered was 1 ml/kg.

The suspending medicine was used as placebo. During experimentalperformance, the arterial blood pressure was continuously recorded 30min. before (basal values) and up to 8 hours after drug administration.

Lercanidipine and (S)-lercanidipine induced a dose-related decrease inarterial blood pressure. The ED₂₅ values (dose inducing 25% decrease inDBP at peak effect) were evaluated by linear regression analysis andsummarized in the Table 4.

                  TABLE 4                                                         ______________________________________                                        Compound          ED.sub.25 (mg/kg) 95% C.L.                                  ______________________________________                                        Lercanidipine     0.9 (0.5 ÷ 1.6)                                         (S)-Lercanidipine 0.4 (0.3 ÷ 0.7)                                         (R)-Lercanidipine >>30                                                        ______________________________________                                         C.L. = Confidence limits                                                 

The (S)-Lercanidipine enantiomer exerted the most potentantihypertensive action. This compound was two fold more efficacious atlowering blood pressure than the racemate. The (R)-enantiomer did notaffect blood pressure up to 30 mg/kg (<10% decrease in DBP).

Example 11A High Temperature Stress Stability

Lercanidipine hydrochloride hemihydrate, prepared according to theprocedure described in U.S. Pat. No. 4,705,797, was compared withanhydrous lercanidipine prepared according to Example 3, to determinethe stress stability of the compounds. The two samples, lercanidipinehydrochloride hemihydrate, and anhydrous lercanidipine hydrochloridewere heated, in the light, at 100° C. for 48 hours. The samples weretested at the following times; 0 hours, 24 hours, and 48 hours. Thepurity of the lercanidipine was checked by HPLC analysis under thefollowing conditions:

Column: m-Bondapak C-18 (Waters), particle size 10 mm, 300×3.9 mm i.d.

Eluant: CH₃ CN (61%):0.15M NaClO₄ aqueous solution at pH 3 (adjustedwith HClO₄) (39%; v/v)

Elution: isocratic

Flow: 1.5 mL/min

Temp. 25° C.

Detector: UV (240 nm)

Attenuation: 0.05 AUFS

The results, expressed as assay % of the compound present, based on anHPLC analysis, are shown in Table 5 below:

                  TABLE 5                                                         ______________________________________                                        Stress Stability at 100° C.                                                       Assay % by HPLC                                                    Compound  0 hr         24 hrs  48 hrs                                         ______________________________________                                        Anhydrous 99.74        99.36   99.01                                          Hemihydrate                                                                             99.85        92.35   90.96                                          ______________________________________                                    

From Table 5, it can be seen that the anhydrous form of lercanidipinehydrochloride exhibited superior stability over the hydrated form.

Example 11B Water Content at 75% Relative Humidity

Samples of anhydrous lercanidipine hydrochloride and lercanidipinehydrochloride hemihydrate were placed in open polyethylene bags inflasks and heated, in the dark, at 60° C. and 75% relative humidity. Thesamples were checked for hygroscopicity by determination of the watercontent using the Karl-Fisher method. The samples were tested at 8 and15 days.

Two additional samples of the anhydrous lercanidipine hydrochloride andlercanidipine hydrochloride hemihydrate were placed in open polyethylenebags in flasks and heated, in the dark, at 40° C. and 75% relativehumidity. The samples were tested as described above at 8 and 15 days.The results of these tests are shown in Table 6.

                  TABLE 6                                                         ______________________________________                                        Water Content at 75% Relative Humidity                                                       Water                                                                         Content                                                                     0 days   8 days  15 days                                         ______________________________________                                        60° C.                                                                          Anhydrous 0.28       0.85  0.77                                               Hemihydrate                                                                             1.42       4.00  4.04                                      40° C.                                                                          Anhydrous 0.28       0.30  0.32                                               Hemihydrate                                                                             1.42       3.14  3.05                                      ______________________________________                                    

From Table 6, it can be seen that the anhydrous lercanidipine has a muchlower tendency to absorb water than the hemihydrate form. The lack ofhygroscopicity is advantageous because the anhydrous compound willremain unchanged. Thus, it will be easier to handle during thepreparation and dispensing of pharmaceutical formulations.

The invention has been described above by reference to preferredembodiments but, as those skilled in the art will appreciate, manyadditions, omissions and modifications are possible all within the scopeof the claims below.

We claim:
 1. A method for preventing, retarding, or reducingatherosclerotic lesions or atherosclerotic degradation of arterial wallsin a patient in need thereof;said method comprising, administering to amammal in need of said prevention, retardation, or reduction, at leastone compound having the formula I: ##STR5## wherein: Ph is phenyl, Aris: 2-nitrophenyl, 3-nitrophenyl, 2,3-dichlorophenyl orbenzofurazan-4-yl A is a branched chain alkylene radical having from 2to 6 carbon atoms, R is a straight or branched chain alkyl radicalhaving from 1 to 6 carbon atoms, optionally mono-substituted by analkoxy substituent having from 1 to 6 carbon atoms, R₁ is hydrogen,hydroxy, or an alkyl radical having from 1 to 4 carbon atoms, R₂ ishydrogen, or methyl;or an enantiomer of a compound having formula I; ahydrated compound having formula I; a solvated compound having formulaI; a pharmaceutically acceptable acid addition salt, or any of theforegoing.
 2. The method according to claim 1, wherein the compoundhaving formula I is an R-enantiomer.
 3. The method according to claim 1,wherein the compound having formula I is an S-enantiomer.
 4. The methodaccording to claim 1, wherein R is methyl, Ar is 3-nitrophenyl, R₁ ismethyl and R₂ is hydrogen.
 5. The method according to claim 1, wherein Ais --C(CH₃)₂ --CH₂ -- or --CH₂ --C(CH₃)₂ --CH₂ --.
 6. The methodaccording to claim 1, wherein the compound having formula I is methyl1,1,N-trimethyl-N-(3,3-diphenylpropyl)-2-aminoethyl1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)pyridine-3,5-dicarboxylate ora pharmaceutically acceptable acid addition salt thereof.
 7. The methodaccording to claim 6, wherein the compound having formula Iis:R-(-)-methyl 1,1,N-trimethyl-N-(3,3-diphenylpropyl)-2-aminoethyl1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)pyridine-3,5-dicarboxylate. 8.The method according to claim 6, wherein the compound having formula Iis:S-(+)-methyl 1,1,N-trimethyl-N-(3,3-diphenylpropyl)-2-aminoethyl1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)pyridine-3,5-dicarboxylate. 9.The method according to claim 1, wherein the dosage of said compound isfrom 0.1 to about 400 mg, per day.
 10. The method according to claim 9,wherein the dosage of said compound is from about 1 to about 200 mg, perday.
 11. The method according to claim 10, wherein the dosage of saidcompound is from about 0.5 to about 100 mg, per day.
 12. The methodaccording to claim 1, wherein said mammal in need of said treatmentsuffers from hypertension.
 13. The method according to claim 1 whereinsaid mammal in need of said treatment does not suffer from hypertension.14. A method for the treatment of atherosclerosis, said methodcomprising, administering to a patient, in need of such treatment, acompound having the formula I: ##STR6## wherein: Ph is phenyl,Ar is:2-nitrophenyl, 3-nitrophenyl, 2,3-dichlorophenyl or benzofurazan-4-yl, Ais a branched chain alkylene radical having from 2 to 6 carbon atoms, Ris a straight or branched chain alkyl radical having from 1 to 6 carbonatoms, optionally mono-substituted by an alkoxy substituent having from1 to 6 carbon atoms, R₁ is hydrogen, hydroxy, or an alkyl radical havingfrom 1 to 4 carbon atoms, R₂ is hydrogen, or methyl; or an enantiomer ofa compound having formula I; a hydrated compound having formula I; asolvated compound having formula I; a pharmaceutically acceptable acidaddition salt, or any of the foregoing.
 15. The method according toclaim 14, wherein the compound having formula I is an R-enantiomer. 16.The method according to claim 14, wherein the compound having formula Iis an S-enantiomer.
 17. The The method according to claim 14, wherein Ris methyl, Ar is 3-nitrophenyl, R₁ is methyl and R₂ is hydrogen.
 18. Themethod according to claim 14, wherein A is --C(CH₃)₂ --CH₂ -- or --CH₂--C(CH₃)₂ --CH₂ --.
 19. The method according to claim 14, wherein thecompound having formula I is methyl1,1,N-trimethyl-N-(3,3-diphenylpropyl)-2-aminoethyl1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl) pyridine-3,5-dicarboxylate ora pharmaceutically acceptable acid addition salt thereof.
 20. The methodaccording to claim 17, wherein the compound having formula Iis:R-(-)-methyl 1,1,N-trimethyl-N-(3,3-diphenylpropyl)-2-aminoethyl1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)pyridine-3,5-dicarboxylate.21. The method according to claim 17, wherein the compound havingformula I is:S-(+)-methyl1,1,N-trimethyl-N-(3,3-diphenylpropyl)-2-aminoethyl1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)pyridine-3,5-dicarboxylate.22. The method according to claim 14, wherein the dosage of saidcompound is from 0.1 to about 400 mg, per day.
 23. The method accordingto claim 22, wherein the dosage of said compound is from about 1 toabout 200 mg, per day.
 24. The method according to claim 23, wherein thedosage of said compound is from about 0.5 to about 100 mg, per day. 25.A method for reducing the migration of cells, reducing build up ofconnective tissues, or preventing lipid accumulation in the bloodvessels of a mammal in need of said treatment comprising administeringto said mammal from 0.1 to about 400 mg, per day, of a compound havingFormula I: ##STR7## wherein: Ph is phenyl,Ar is: 2-nitrophenyl,3-nitrophenyl, 2,3-dichlorophenyl or benzofurazan-4-yl, A is a branchedchain alkylene radical having from 2 to 6 carbon atoms, R is a straightor branched chain alkyl radical having from 1 to 6 carbon atoms,optionally mono-substituted by an alkoxy substituent having from 1 to 6carbon atoms, R₁ is hydrogen, hydroxy, or an alkyl radical having from 1to 4 carbon atoms, and R₂ is hydrogen, or methyl.